Ongoing studies of the ensymology of complex polyketide natural product biosynthesis will be continued and extended, with focus on the macrolide antibiotics erythromycin (1), methymycin (2), and tylosin (3), as well as the antitumor metabolite epothilone (4). Each of these metabolites is assembled by exceptionably large, multifunctional, modular proteins known as polyketide synthases (PKSs) that are closely related to fatty acid synthases, both biochemically and genetically. In addition, epothilone synthase contains additional catalytic activities belonging to the class of non-ribosomal peptide synthetases (NRPSs). A combination of chemical, enzymological, and molecular genetic techniques this being used to elucidate the molecular basis for the programming of the complex series of reactions responsible for polyketide chain elongation. The emphasis in this work is on the elucidation of the mechanisms of multi-step, enzyme-catalyzed transformations leading to formation of biologically important metabolites. It is expected that the results of these studies will be broadly applicable not only to the understanding of polyketide and other natural product biosynthetic processes in general, but will provide fundamental insights into how catalysis and molecular recognition control both product specificity and molecular diversity in Nature. 1) Deoxyerythronolide B synthase (DEBS) is a modular PKS that catalyzes the formation of 6-deoxyerythronolide B (5), the parent aglycone of erythromycin A. Individual modules of the DEBS protein, responsible for catalysis of a single round of polyketide chain elongation and functional group modification, can be expressed in E. coli. These modules will be used to study the biochemical basis for the specificity and selectivity of individual catalytic domains, particularly the ketosynthase (KS) domains that mediate the key polyketide chain-building decarboxylative condensation reaction. 2) The methymycin and tylosin PKSs have intriguing similarities and differences to the well-studied DEBS system. Individual modules of the methymycin/picromycin and tylactone PKSs will be expressed in E. coli in order to investigate their biochemical function and substrate specificity. 3) The EpoA protein, the loading module for the epothilone hybrid PKS/NRPS, will be expressed in E. coli in order to study the EpoA-catalyzed conversion of malonyl-CoA to acetyl-S-EpoA, the substrate for the NRPS module EpoB.